Resistance to thyroid hormone (RTH) is a human syndrome characterized by abnormal regulation of the hypothalamic-pituitary-thyroid axis (HPT axis) leading to inappropriate TSH secretion and elevated serum thyroid hormone levels. A central role for thyroid hormone receptor-beta (TR-beta) gene in thyroid hormone negative regulation of the HPT axis was established by finding TR-13 gene mutations in patients with RTH and by deleting the TR-beta isoforms in mice. Despite years of study, however, the mechanism of thyroid hormone inhibition of the HPT axis remains unclear. In normal thyroid hormone action, TR-a interacts with co-repressor molecules in the absence of ligand (T3) and co-activator molecules in the presence of T3 to stimulate gene expression. In contrast, the mechanism of negative regulation by thyroid hormone is less clear. Understanding how the same receptor and associated cofactors both stimulates and inhibits gene expression would enhance our understanding of transcriptional signaling processes and may provide the basis for discovery of novel signaling pathways. To accomplish this goal three specific aims are proposed: A.1 To elucidate the mechanism of T3 inhibition by using a mouse thyrotroph (TSH-secreting) cell line (TaT1.1). Using a variety of methods, a subclone of the TaT1 cell line (TaT1.1) will be used to understand the nature of the protein complexes bound to the TSH-beta nTRE and their functional significance. A.2 To determine the direct role of co-activators in TR-beta mediated T3 inhibition in vivo. A point mutation was introduced into helix 12 (E457A) of the mouse TR-beta locus (E457A knock-in). This mutation specifically interferes with co-activator binding without affecting co-repressor or ligand (T3) binding. The effect of this mutation on both positive and negative T3 regulation will be determined in vivo. A.3 To determine the direct role of co-repressors in TR-beta mediated T3 inhibition in vivo. A point mutation (I280M) will be introduced into mouse TR-beta locus (I280M knock-in). This mutation specifically interferes with co-repressor binding without affecting either co-activator or ligand binding. The effect of this mutation on both positive and negative T3 regulation will be determined in vivo. Study of other animal models, where co-repressor function has been altered, is also planned in this aim.